The type,amount, location,and energy transfer properties of the carotenoid in reaction centers from Rhodopseudomonas sphaeroides

Analysis of photosynthetic reaction centers from Rhodopseudomonas sphaeroides strains 2.4.1 and Ga shows that each contains approx. 1 mol of a specific carotenoid per mol of reaction center. In strain 2.4.1. the carotenoid is spheroidene (1-methoxy-3,4-didehydro-1,2,7′,8′-tetrahydro-ψ,ψ-carotene); in strain Ga, it is chloroxanthin (1-hydroxy-1,2,7′,8′-tetrahydro-ψ,ψ-carotene). The carotenoid is bound to the same pair of proteins as are the bacteriochlorophylls and bacteriopheophytins of the reaction center. This binding induces strong circular dichroism in the absorption bands of the carotenoid. The carotenoid is close enough to the other pigments of the reaction center so that light energy transfers efficiently from the carotenoid to the bacteriochlorophyll, sensitizing bacteriochlorophyll fluorescence. The fluorescence polarization spectrum of the reaction centers shows that the transition vectors for the visible absorption bands of the carotenoid lie approximately parallel to the 600 nm ($\text{Q}{}_{\mathrm{<mtext>x</mtext>}}$) transition of the bacteriochlorophyll complex.     

The triplet exciton of chlorophyll a and carotenoid in solution and in photosynthetic antenna proteins

We have observed the development and decay of triplet excitons formed in the ‘antenna’ chlorophyll $\text{a}\text{b}$ protein complex by high-intensity laser excitation. The carotenoid triplet (³Car) appeared 5 ns after excitation in the protein isolation, commonly termed CP-II; the risetime in a larger antenna particle, called LHC (light-harvesting complex) was 12 ns. The quantum yield of ³Car in CP-II decreased 11-fold as intensity was increased from 10¹⁶ to 2 · 10¹⁷ photons/cm² per pulse. The effect is attributed to exciton annihilation during the initial period of triplet formation. Above 5 · 10¹⁶ photons/cm² per s, the ³Car lifetime decreases substantially from its low intensity value of 8.7 μs. A comparison of the transient absorption spectrum of CP-II with those of chlorophyll and carotenoid in vitro indicates that ‘trapped’ chlorophyll triplets formed at high intensities. We present a simple model of destructive interaction between ³Car and chlorophyll triplets which is compatible with the observed increased rate of ³Car decay. Indirect evidence suggests similar effects occur in LHC.     

Binding properties of 23S,25-dihydroxyvitamin D3: an in vivo metabolite of vitamin D3

23$\text{S}$,25-Dihydroxyvitamin D₃ was isolated from the plasma of vitamin D₃-toxic pigs. An ultraviolet absorbance spectrum confirmed its purity. The configuration of the 23-hydroxyl group was determined to be S by comparison of the natural product with synthetic 23$\text{R}$,25- and 23$\text{S}$,25-dihydroxyvitamin D₃ by high-pressure liquid chromatography. The affinity of both 23$\text{S}$,25- and 23$\text{R}$,25-dihydroxyvitamin D₃ for the plasma vitamin D binding protein was similar to vitamin D₃. Thus, with respect to the plasma vitamin D binding protein, 23$\text{S}$,25-dihydroxyvitamin D₃ is the least potent, naturally-occurring, dihydroxylated vitamin D₃ metabolite known.     

Toluene dioxygenase: purification of an iron-sulfur protein by affinity chromatography

Toluene dioxygenase, from $\text{Pseudomonas}\text{putida}$, oxidizes toluene to (+)-$\text{cis}$-1(S),2(R)-dihydroxy-3-methylcyclohexa-3,5-diene. The oxygenase-component of this multienzyme system was purified to homogeneity by a two-step procedure that utilized affinity and ion exchange chromatography. The purified enzyme would oxidize toluene only in the presence of NADH, ferrous iron and partially purified preparations of NADH cytochrome c reductase and an iron-sulfur protein (ferredoxin_TOL). Spinach NADPH cytochrome c reductase and NADPH could substitute for the $\text{Pseudomonas}$ reductase and NADH. The molecular weight of the oxygenase-component was determined to be 151,000 and polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate indicated that the enzyme is composed of two subunits with molecular weights of 52,500 and 20,800. The absorption spectrum showed maxima at 550 (Shoulder), 450, 326 and 278 nm and preliminary experiments have indicated the presence of 2 gram atoms of iron and 2 gram atoms of acid-labile sulfur per mole of protein. The results indicate that the oxygenase-component of the toluene dioxygenase enzyme system is an iron-sulfur protein that has been designated ISP_TOL.     

Isolation and properties of the cytochrome B-C1 complex from Rhodopseudomonas sphaeroides

A highly purified and active cytochrome $\text{b}$-$\text{c}$₁ complex has been isolated from the chromatophores of the photosynthetic bacteria $\text{Rhodopseudomonas}\text{sphaeroides}$ R-26, through steps of Triton X-100 solubilization, salt fractionation and calcium phosphate column chromatography. The isolated enzyme complex catalyzes fully antimycin A sensitive oxidation of ubiquinol by cytochrome $\text{c}$ with a turnover number of 1500 per minute at 23° based on cytochrome $\text{c}$₁. It contains 8.3 nmoles of cytochromes $\text{b}$ and $\text{c}$₁ per mg protein and shows four polypeptides in the sodium dodecylsulfate polyacrylamide gel electrophoresis.     

Purification and characterization of cytochrome C3 (Mr 26,000) isolated from Desulfovibriodesulfuricans Norway strain

An acidic cytochrome c (P_i = 4.8) has been purified from $\text{Desulfovibrio}\text{desulfuricans}$ Norway. Its molecular weight was estimated to be 26,000 but a monomeric form of 13,500 molecular weight has been obtained. The comparison of its amino acid composition and N terminal sequence has characterized this cytochrome as a new cytochrome, different from cytochrome c₃ (Mr 13,000) and cytochrome c_553(550) studied in the same organism. Its optical spectrum was similar to cytochrome c₃ (Mr 13,000) accordingly it has 4 haems per subunit. The absence of absorption at 695 nm indicates that two histidine residues are implicated as fifth and sixth ligand for haem iron. This new cytochrome is homologous to the cytochrome C₃ (Mr 26,000) previously described for $\text{Desulfovibrio}\text{gigas}$ and $\text{Desulfovibrio}\text{vulgaris}$.     

Antenna function of a chlorophyll ab protein complex of Photosystem I

The functional role of a chlorophyll $\text{a}\text{b}$ complex associated with Photosystem I (PS I) has been studied. The rate constant for P-700 photooxidation, K_P-700, which under light-limiting conditions is directly proportional to the size of the functional light-harvesting antenna, has been measured in two PS I preparations, one of which contains the chlorophyll $\text{a}\text{b}$ complex and the other lacking the complex. K_P-700 for the former preparation is half of that of the preparation which has the chlorophyll $\text{a}\text{b}$ complex present. This difference reflects a decrease in the functional light-harvesting antenna in the PS I complex devoid of the chlorophyll $\text{a}\text{b}$ complex. Experiments involving reconstitution of the chlorophyll $\text{a}\text{b}$ complex with the antenna-depleted PS I preparation indicate a substantial recovery of the K_P-700 rate. These results demonstrate that the chlorophyll $\text{a}\text{b}$ complex functions as a light-harvesting antenna in PS I.     

Binding of cytochrome c to the cytochrome bc1 complex (complex III) and its subunits cytochrome c1 and b1.

Cytochrome $\text{c}$₁, the electron donor for cytochrome $\text{c}$, is a subunit of the mitochondrial cytochrome $\text{bc}$₁ complex (complex III, cytochrome $\text{c}$ reductase). To test if cytochrome $\text{c}$₁ is the cytochrome $\text{c}$-binding subunit of the $\text{bc}$₁ complex, binding of cytochrome $\text{c}$ to the complex and to isolated cytochrome $\text{c}$₁ was compared by a gel-filtration method under non-equilibrium conditions (a $\text{bc}$₁ complex lacking the Rieske ironsulfur protein was used; von Jagow et al. (1977) Biochim. Biophys. Acta $\text{462}$, 549–558). The approximate stoichiometries and binding affinities were found to be very similar. Binding of cytochrome $\text{c}$ to isolated cytochrome $\text{b}$ which is another subunit of the reductase was not detectable by the gel-filtration method. Further, the same lysine residues of cytochrome $\text{c}$ were shielded towards chemical acetylation in the complexes $\text{c}\text{:}\text{c}$₁ and $\text{c}\text{:}\text{bc}$₁. From this we conclude that the same surface area of cytochrome $\text{c}$ is in direct contact with cytochrome $\text{bc}$₁ and with cytochrome $\text{c}$₁ in the respective complexes and that therefore cytochrome $\text{c}$ is most probably the structural ligand for cytochrome $\text{c}$ in mitochondrial cytochrome $\text{c}$ reductase.     

A cobalt porphyrin containing protein reducible by hydrogenase isolated from Desulfovibrio desulfuricans (Norway)

A cobalt-porphyrin containing protein has been isolated from the sulfate-reducer $\text{Desulfovibrio desulfuricans}$ (Norway). This violet-colored protein has a molecular weight of approx. 13,000 daltons and contains 1 cobalt atom/molecule. The apo-protein was estimated to contain 104 amino-acid residues giving a molecular weight of 11,000 daltons. The UV-visible absorption spectrum of the protein exhibiting maxima at 588,418 and 280 nm with a shoulder at 550 nm is characteristic of metalloporphyrin proteins. The molar extinction coefficients of the cobalt-protein at 588, 418 and 280 nm are 31,330 , 64,670 and 17,200 respectively and its absorbance ratio $\text{A}{}_{280}\text{A}{}_{588}$ is 0.54. The protein is reduced by dithionite giving a blue-colored reduced form. Important spectral modifications of the chromophore occurred during the reduction including a shift of the Soret peak from 418 to 381 nm and a shift of the α band in the opposite direction from 588 to 593.5 nm. The Co-protein was slowly reduced by the hydrogenase from $\text{D.desulfuricans}$ under hydrogen in the presence of cytochrome C₃. The reported data suggest that the redox states of the cobalt center of this new electron carrier correspond to the Co(III) and Co(II) states.     

1,25-Dihydroxyvitamin D3-glycoside: Identification of a calcinogenic principle of solanum malacoxylon

The water soluble calcinogenic factor present in the plant $\text{Solanum}$$\text{malacoxylon}$ is partially purified by selective extraction and chromatography on silicic acid and then hydrolyzed with a mixed preparation of glycosidases from the sea worm, $\text{Charonia}$$\text{lampus}$. Hydrolysis produces a chloroform soluble factor with biologic characteristics of 1,25-dihydroxyvitamin D₃ (1,25-(OH)₂D₃), the hormonal form of vitamin D. Purification of this factor is accomplished by chromatography on Sephadex LH-20, silicic acid, and Celite columns, yielding 3 μg of active material. During the isolation, bioactivity (as assessed by the ability of fractions to compete with labeled 1,25-(OH)₂D₃ for binding to a specific intestinal receptor protein) migrates exactly with authentic tritiated 1,25-(OH)₂D₃. The purified factor has an ultraviolet absorption spectrum identical to that of 1,25-(OH)₂D₃ and analysis via direct probe mass spectrometry yields a parent molecular ion of m/e 416 and a fragmentation pattern indistinguishable from synthetic 1,25-(OH)₂D₃ hormone. We therefore conclude that the vitamin D-like principle in $\text{Solanum}$$\text{malacoxylon}$ is a sterol-glycoside which contains the 1,25-(OH)₂D₃ molecule as its active sterol component.     

Isolation of QP-C and reconstitution of the QH2-c reductase

A ubiquinone protein, QP-C, which acts in the cytochrome $\text{b}\text{-}\text{c}$₁ region has been solubilized. The isolated QP-C shows one band of molecular weight 15,000 in polyacrylamide gel electrophoresis in sodium dodecyl sulfate and isoelectric focusing at the isoelectric point of pH 3.6. QP-C reconstitutes with the QP-C deficient $\text{b}\text{-}\text{c}$₁ complex to restore the OH₂-cytochrome $\text{c}$ activity and recover the EPR signal of the complex.     

A new membrane iron-sulfur flavoprotein of the mitochondrial electron transfer system the entrance point of the fatty acyl dehydrogenation pathway?

An iron-sulfur (FeS) protein has been purified from beef heart mitochondria by following its EPR signal after reduction, which is characteristic of a ferredoxin-type FeS protein (g_x = 1.886; g_y = 1.939; g_z = 2.086). The signal intensity corresponds to one unpaired spin for 4 to 5 Fe atoms. The light absorption spectrum indicates the presence of flavin. Fe, labile S, and FAD are released by acid at a ratio of approximately 4:4:1. Neither prosthetic group of the protein is reduced by NADH, NADPH, succinate, glycerol-3-phosphate or dihydroorotate. The FeS group is, however, reduced with a half-time of ～5 msec, when the protein is mixed with an equivalent amount of electron transferring flavoprotein (ETF) of the β-oxidation cycle, prereduced with an acyl CoA dehydrogenase and a saturated fatty acyl CoA. In the presence of the two added flavoproteins the behavior of the flavin of the FeS flavoprotein could not be determined. Complexes I–III are not reduced by reduced ETF under analogous conditions. The low field EPR resonance [“center 5”, Ohnishi $\text{et al}$. (1972), Biochem. Biophys. Res. Commun. $\text{46}$, 1631–1638] of the protein is readily observed in whole tissue, mitochondria and sonic fragments from all species we have examined. Therefore, the protein appears to be a universal constituent of mitochondrial electron transfer systems.     

Evidence for a novel circulating alpha 1 protein in tumour-bearing rats. Differentiation from acute phase alpha 1 proteins and from alpha 1 fetoprotein

It has been found in this study that the serum from rats bearing a transplanted dibenzanthracene-induced tumour ($\text{RD}\text{3}$), has a high concentration of alpha₁ proteins compared with normal rat serum. These alpha₁ proteins have been isolated by an immunoabsorption method and have been compared by immunological methods with the acute phase alpha₁ proteins isolated by the same method from the serum of rats presenting an inflammatory reaction. It has been found that the isolated $\text{RD}\text{3}$ alpha₁ proteins were composed of two major proteins: one of these corresponded to an inflammatory protein, the alpha₁-AP-globulin. The other may be a new protein, as it is absent from the serum of rats with an acute phase inflammatory reaction and nor does it correspond to alpha₁ feto-protein, a carcino-embryonic protein presenting the same electrophoretic mobility.     

Rapid incorporation of the human neutrophil plasma membrane cytochrome b into phagocytic vacuoles

Phagocytic vacuoles containing lgG coated latex particles were isolated from human neutrophils by floatation. The absorbance spectrum of the cytochrome $\text{b}$ was associated with the vacuoles within 10 sec of particle uptake and the vacuolar concentration increased little thereafter. In contrast, the cytoplasmic granule proteins myeloperoxidase and vitamin B₁₂ binding protein associate with the vacuoles more slowly. The addition of dithionite to intact cells rapidly reduces most of the cytochrome $\text{b}$, whereas only a small proportion of the myeloperoxidase, which is located intracellularly, is reduced in the absence of detergent. Most of the cytochrome $\text{b}$ appears to be localised in the neutrophil plasma membrane.     

Oxidation-reduction properties of the electron acceptors of Photosystem II I. Redox titration of the flash-induced carotenoid band shift,of C550 and of the variable fluorescence yield in spinach chloroplasts

Redox titration of the electrochromic carotenoid band shift, detected at 50 μs after a saturating actinic flash, in spinach chloroplasts, shows that only one electron acceptor in Photosystem II participates in a transmembrane primary electron transfer. This species, the primary quinone acceptor, Q, shows only one midpoint potential (E_m,7.5) of approx. 0 V and is undoubtedly equivalent to the fluorescence quencher, Q_H. A second titration wave is observed at low potential ($\text{E}{}_{\mathrm{<mtext>m</mtext><mtext>,7.5</mtext>}}\text{? ? 240}\text{mV}$) and at greater than 3 ms after a saturating actinic flash. This wave has an action spectrum different from that of Photosystem II centers containing Q and could arise from a secondary but not primary electron transfer. A low-potential fluorescence quencher is observed in chloroplasts which largely disappears in a single saturating flash at ? 185 mV and which does not participate in a transmembrane electron transfer. This low-potential quencher (probably equivalent to fluorescence quencher, Q_L) and Q are altogether different species. Redox titration of C550 shows that if electron acceptor Q_β is indeed characterized by an E_m,7 of + 120 mV, then this acceptor does not give rise to a C550 signal upon reduction and does not participate in a transmembrane electron transfer. This titration also shows that C550 is not associated with Q_L.     

Nondiscrimination of RNA viral message in binding to 30S ribosomes derived from T4 phage infected Escherichia coli

The effect of T4 phage on ribosomes in terms of their ability to bind RNA viral template is examined. It is found that the 30S subunits of T4 ribosomes bind MS2 RNA as efficiently as do the subunits of uninfected $\text{E. coli}$ ribosomes. On the other hand, analyses of the formation of 70S initiation complex, presumably from MS2 RNA-30S ribosome complex, using both labeled MS2 RNA and initiator tRNA, reveal that T4 ribosomes are only about half as active as $\text{E. coli}$ ribosomes. The latter phenomenon has been reported previously. These results suggest that, following T4 infection, ribosomes are modified in such a way that the attachment of fMet-tRNA_f to MS2 RNA-30S subunit complex is impaired.     

Isolation and characterization of a major chlorophyll ac2 light-harvesting protein from a Chroomonas species (Cryptophyceae)

Three chlorophyll-protein complexes of a Chroomonas species (Cryptophyceae) have been separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis. The two bands at 100 and 42 kDa are Complex I (CP I) and Complex IV (CP IV), the ubiquitous chlorophyll a-proteins associated with Photosystems I and II, respectively. The third 55 kDa band, which had two peptide subunits (24 and 20 kDa), contained both chlorophyll a and chlorophyll c₂ in a molar ratio of 1.4 chlorophyll a to 1 chlorophyll c₂ ($\text{chlorophyll}\text{a}\text{chlorophyll}\text{c}{}_2$ ratio in whole cells = 4). A chlorophyll $\text{a}\text{c}{}_2$ fraction with similar spectral and electrophoretic properties was isolated by digitonin-sucrose density gradient centrifugation. This fraction had no photochemical activity and contained only a single carotenoid species with absorbance maxima in methanol at 424, 448 and 476 nm. Efficient energy transfer from chlorophyll c₂ to chlorophyll a occurred in the complex.     

Protein rotation and chromophore orientation in reconstituted bacteriorhodopsin vesicles

Bacteriorhodopsin has been reconstituted into lipid vesicles with dipalmitoyl and dimyristoyls phosphatidylcholine. Circular dichroism (CD) measurements show that the proteins are in a monomeric state above the main lipid phase transition temperature (T_c), 41 and 23°C for dipalmitoyl and dimyristoyl phosphatidylcholine, respectively. Below T_c, the CD spectrum is the same as that found for the purple membrane. The latter result implies that the orientation of the chromophore at these temperatures is most likely the same as in the purple membrane ($\text{70° ± 5°}$ from the normal to the membrane plane).Transient dichroism measurements show that below T_c the proteins are immobile, while above this temperature protein rotation around an axis normal to the plane of the membrane is occurring. In addition, from the data the angle of the chromophore for the rotating proteins with respect to the rotational diffusion axis can be calculated. This angle is found to be $\text{30° ± 3°}$ and $\text{29° ± 4°}$ in dimyristoyl phosphatidylcholine and dipalmitoyl phosphatidylcholine, respectively. This is considerably smaller than the value of $\text{70° ± 5°}$ for the natural biomembrane. A reversible reorientation of the chromophore above and below the respective main T_c transition temperature could explain the change of angle observed provided that all the molecules rotate above T_c.     

Crystallization of and preliminary X-ray diffraction data for TET-repressor and the TET-repressor-tetracycline complex

The TET-repressor encoded by the transposon Tn10 has been crystallized along with the repressor-tetracycline complex. Both crystals belong to the space group P4₃2₁2 (or P4₁2₁2) with cell dimensions $\text{a = b = 74.3(1)}\text{A}\text{?}\text{, c = 94.2(2)}\text{A}\text{?}\text{and}\text{a = b = 73.3(1)}\text{A}\text{?}\text{, c = 94.6(2)}\text{A}\text{?}$ for the free and complexed repressor, respectively. There is one molecule of molecular weight 23,000 per asymmetric unit, and the biologically active dimer therefore consists of two identically formed subunits which are related by a crystallographic 2-fold axis. This isomorphism of TET-repressor and its tetracycline complex suggests that only minor, subtle changes in structure trigger binding to or release of the operator. The crystals of the native protein permit X-ray data collection to 3.2 Å and those of the complexed repressor to 2.8 Å.